【作者】 潘哲超；

【导师】 冯洁；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2010， 博士

【摘要】 植物青枯病是由茄科雷尔氏菌(Ralstonia solanacearum)引起的世界性重要细菌性病害,寄主范围极为广泛,可侵染54个科的450余种植物;不同地理起源的青枯菌在与其寄主长期协同进化的过程中,演化出明显的生理分化或菌系多样性。传统的分类将青枯菌划分为5个生理小种和5个生化变种,但传统的分类框架不能反映青枯菌的遗传进化及与地理起源的相关性。Fegan和Prior(2005)共同提出了演化型分类框架用于描述青枯菌种以下的分类,得到了国际上的广泛认可。我国迄今为止尚未开展青枯菌演化型分类鉴定的相关研究,本研究采用国际上最新的青枯菌演化型分类框架,对我国青枯菌进行了遗传多样性分析,同时基于遗传多样性分析结果,以遗传背景相近但寄主范围存在明显差异的菌株为研究对象,运用抑制差减杂交技术和蛋白质双向电泳技术筛选寄主适应性基因,旨在探究中国青枯菌菌系生物学特性、进化关系及致病力分化,从而为改进育种策略、发展分子检测技术及青枯菌致病机制研究奠定基础。1.青枯菌遗传多样性研究本研究分别在演化型和序列变种两个分类水平上对中国青枯菌群体进行了种以下分类研究。在演化型分类水平上,揭示出来自13个省、18个寄主的286个参试菌株全部属于青枯菌演化型I和II型,即亚洲分支和美洲分支菌株。在序列变种分类水平上揭示出中国青枯菌群体具有丰富的遗传多样性,存在着亚洲分支的10个序列变种及美洲分支的1个序列变种。其中亚洲分支菌株中的序列变种33、44和48为国际上首次鉴定出的新序列变种。2.青枯菌致病力分化研究在序列变种鉴定过程中,发现了在致病力分化上具有重要意义的来自马铃薯的Po82菌株,与寄主范围差异很大的香蕉青枯菌及NPB(not pathogenic to banana)菌株同属序列变种4。AFLP聚类分析的结果显示:Po82在亲缘关系上与NPB菌株更近;SDS-PAGE分析Po82胞外泌出蛋白谱带类型与香蕉青枯菌菌株更为接近。致病力鉴定结果表明:Po82能够侵染茄科植物的番茄、茄子及马铃薯,且兼具对香蕉的致病力,是迄今为止发现的第一株可对香蕉致病的来自马铃薯的菌株,从而表明Po82是一个进化地位上极为独特的菌株。3. SSH筛选青枯菌寄主适应性基因利用抑制性差减杂交技术,构建了4个不同类型的差减文库:马铃薯菌株(Po82)/香蕉青枯菌(2号小种,RUN92)、马铃薯菌株(Po82)/NPB菌株(RUN292)、姜青枯菌(4号小种,Z1)/广寄主菌株(1号小种,GMI1000)及桑青枯菌(5号小种,M7/M2/M3)/广寄主菌株(1号小种,GMI1000),筛选获得了不同差减库的特异性核苷酸片段,其功能主要涉及跨膜蛋白、转录调控蛋白、信号肽、假设蛋白以及未发现同源性的片段等几大类。桑青枯菌的3个差减文库中均含有大量携带IS插入元件IS1420和ISRso15的差异性片段,在GMI1000(1号小种)及其它已测序的小种中均未发现IS1420元件,在GMI1000中仅存在2个ISRso15元件,推断其可能与桑青枯菌寄主适应性有关。此外,基于差减片断MG67成功地建立了桑青枯菌(5号小种)的PCR检测体系。4.桑青枯菌Ш型分泌系统突变株的构建及其泌出蛋白分析通过同源置换法构建了GMI1000△popP1和桑青枯菌M7△hrpB突变株,利用蛋白质双向电泳技术,分析比较了M7与突变株M7△hrpB泌出蛋白的差异,为进一步筛选桑青枯菌(5号小种)的Ⅲ型效应蛋白奠定了基础。利用演化型及序列变种分类单元对青枯菌遗传多样性的研究,明确了我国青枯菌种以下的分类地位,发现了3个新的序列变种;发现了进化地位十分特殊的Po82菌株可对香蕉致病;桑青枯菌中大量插入子元件的存在可能与其寄主适应性相关;上述研究对青枯菌致病力分化及致病机理研究具有重要意义。更多还原

【Abstract】 As a diverse species complex, Ralstonia solanacearum has developed an extremely broad host range throughout the world, including more than 450 host species representing 54 plant families. Biovar typing and race assessment are methods commonly used for assessing the diversity of R. solanacearum strains. However, recent genetic evidence has indicated that these phenotypically-based schemes are not sufficient to encompass the diversity of strains represented in the species R. solanacearum. Fegan and Prior (2005) proposed a new hierarchical classification scheme to distinguish the genetic diversity within the R. solanacearum species complex. The phylotyping scheme is highly discriminatory, flexible, additive, and should allow better prediction of the properties of strains.Although considerable research has been conducted on bacterial wilt disease in China, less work has been done on the genetic diversity of R. solanacearum species complex. The aim of this study was to use the phylotyping scheme to determine the genetic diversity of R. solanacearum strains from China. Based on the pathogenic properties and phylogenetic relationships among the R. solanacearum strains, suppression subtractive hybridization (SSH) and two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) were then used to isolate host-specific gene. Such understanding allowed identification of biological properties, evolutionary relationships and pathogenicity variation of infra-subspecific groups of strains and result in improving breeding strategies, developing targeted diagnostic tests and studying pathogenesis of R. solanacearum.1. Genetic Diversity of Ralstonia solanacearum Strains from ChinaA survey of bacterial wilt in China collected 286 strains of Ralstonia solanacearum from 17 plant species in 13 Chinese provinces. A phylotype-specific multiplex-PCR showed that 198 isolates belonged to phylotype I (bv3, 4 and 5) and 88 to phylotype II (bv2 and bv1). A phylogenetic analysis examined the partial sequence of the egl and hrpB gene of all strains and the genetic diversity of 95 representatives were reported, demonstrating that Chinese strains are partitioned into phylotype I (Asia) and II (America). Phylotype I strains (historically typed bv3, 4 and 5), had considerable phylogenetic diversity, including 10 different sequevars: seven previously described sequevars 12 to 18 and three new sequevars: 34, 44 and 48.2. Pathogenicity Variation of Ralstonia solanacearum StrainsPo82, a potato strain from Mexico, was resolved into phylotype II sequevar 4, in a group with phylotype II/4NPB and banana Moko disease-causing strains. It was identified as Moko strains’sequevar 4 (MLG25) according to Mmx-PCR. AFLP fingerprinting showed that Po82 was more closely related to NPB than to moko disease-causing strains. However, SDS-PAGE of total cellular protein extracted from Po82 indicated that its protein pattern was more closely related to Moko disease-causing strains than to NPB. Additionally, pathogenicity test revealed that Po82 possess pathogenic traits of both NPB and Moko strains thereby proving that it was a pathogenicity variation strain.3. Differences between Ralstonia solanacearum strains Revealed by Suppression Subtractive HybridizationSSH approach was used to investigate the pathogenic determinants of the R. solanacearum strain. The genome of the R. solanacearum tester strain was subtracted from the genome of the R. solanacearum driver strain, resulting in the identification of subtracted fragments. The majority of the fragment sequences were homologous to Rhs proteins, transmembrane proteins, transcription regulator proteins, signal peptide proteins and hypothetical proteins. Sequence analysis indicated that many fragments in the mulberry strain were disrupted by insertion sequences (IS). A PCR diagnostic test for detection of Ralstonia solanacearum race 5-biovar 5 strains was developed.4. Mutant construction of hrpB gene and Analysis of Type III-Dependent EffectorsA comparative proteome analysis was initiated to investigate the Type III-dependent effectors of Ralstonia solanacearum strain M7 (race5). The mutant of hrpB and popP1 were constructed and subsequently used for pathegenicity and proteome analysis. 2-D IEF/SDS-PAGE allowed the separation of Type III-Dependent Effectors.In summury, an in-deep study of genetic diversity of R. solanacearum strains from China was determined by modern phylotype-sequevar phylogenetic analysis method, Po82, a potato strain was first reported could be pathogenic to banana. Construction of the subtracted DNA library revealed that IS elements may play a role in the evolution of mulberry Ralstonia solanacearum strains. These new findings may provide important clues when studying pathogenicity determinants in the R. solanacearum strains.更多还原